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Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Background: Cytoskeletal degradation occurs in warm renal ischemia and reperfusion and during hypothermia. The purpose of this study was to determine cytoskeletal changes during cold storage preservation injury in renal tubules and to determine whether these changes contribute to the injury., Methods: Isolated canine renal proximal tubules or their primary epithelial cultures were cold-stored in University of Wisconsin solution and reperfused in vitro to simulate renal cold storage preservation injury. Assays for cytoskeletal protein degradation and viability together with biologically active cytoskeletal agents and molecular interventions were used to test this hypothesis., Results: Progressively increasing the cold storage time of isolated renal tubules in University of Wisconsin solution caused proportional disassembly of both ezrin and Na/K adenosine triphosphatase proteins from their cytoskeletal attachments. The sublamellar structural protein, fodrin, was metabolized to products consistent with calpain hydrolysis during preservation. Time-dependent deterioration in tubule membrane function (organic cation transporter-1 transport activity) was observed during cold preservation, and this was mimicked in fresh tubules by chemically induced cytoskeletal disruption with cytochalasin-D treatment. Cold preservation decreased total tubulin content. Taxol slowed this loss by preventing tubulin depolymerization, which improved membrane function and tubule viability. The viability of primary renal epithelial cells was enhanced by overexpression of ezrin (transfection) and diminished with specific ezrin small interfering RNA knockdown during cold preservation injury., Conclusion: Hypothermic storage preservation causes disruption of key cytoskeletal elements in kidney tubules, which contributes causally to the injury at rewarming.

Background: Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury., Methods: Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury., Results: Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0-120 h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion)., Conclusions: This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.

Background: Donation of kidneys from non-heart beating donors (NHBD) is increasingly being used to expand the donor pool. Warm ischemic injury of these kidneys suffered at harvest results in DGF at transplantation. In this study, we used hypothermic continuous machine perfusion preservation to mitigate this injury using two available solutions., Methods: Dog kidneys (beagles) were exposed to 0, 60, or 75 min of in situ warm ischemia (37 degrees C), followed by 24 to 72 hr preservation by machine perfusion with Belzer MPS solution or the UW-solution (Viaspan). Auto-transplantation was performed with immediate contralateral nephrectomy. Survival and renal function (serum creatinine) were evaluated for up to 10 days posttransplant., Results: Both solutions were equally effective for 72 hr machine perfusion preservation of dog kidneys giving 100% survival with only minor renal injury. Both solutions were also equally effective for preservation of kidneys exposed to 60 min of warm ischemia. However, only the UW solution gave reliable preservation (86% survival vs. 25% survival) in kidneys exposed to 75 min of warm ischemia and 24 hr machine perfusion., Conclusion: UW solution used with continuous hypothermic machine perfusion preservation can rescue canine kidneys from severe warm ischemic injury.

Background: In the rodent, ischemic preconditioning (IPC) has been shown to improve the tolerance of the liver to ischemia-reperfusion under normothermic or hypothermic conditions. The aim of the present study was to test this hypothesis in a dog model, which may be more relevant to the human., Methods: Beagle dogs were used in two distinct animal models of hepatic warm ischemia and orthotopic liver transplantation (hypothermic ischemia). IPC consisted of 10 minutes of ischemia followed by 10 minutes of reperfusion. In the first model, livers were exposed to 55 minutes prolonged warm ischemia and reperfused for 3 days (n = 6). In the second model, livers were retrieved and preserved for 48 hours at 4 degrees C in University of Wisconsin solution, transplanted, and reperfused without immunosuppression for 7 days (n = 5). In each model, nonpreconditioned animals served as controls (n = 5 in each group). Also, isolated dog hepatocytes were subjected to warm and cold storage ischemia-reperfusion to model the animal transplant studies using IPC., Results: In the first model (warm ischemia), IPC significantly decreased serum aminotransferase activity at 6 and 24 hours post-reperfusion. After 1 hour of reperfusion, preconditioned livers contained more adenosine triphosphate and produced more bile and less myeloperoxidase activity (neutrophils) relative to controls. In the second model (hypothermic preservation), IPC was not protective. Finally, IPC significantly attenuated hepatocyte cell death after cold storage and warm reperfusion in vitro., Conclusions: IPC is effective in large animals for protecting the liver against warm ischemia-reperfusion injury but not injury associated with cold ischemia and reperfusion (preservation injury). However, the IPC effect observed in isolated hepatocytes suggests that preconditioning for preservation is theoretically possible.

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.

This study was conducted to investigate the effect of starch in the preservation solution during hypothermic machine perfusion (HMP) on endothelial cell and hepatocyte functions in an isolated perfused rat liver model. Livers isolated from male Sprague-Dawley rats were perfused with the University of Wisconsin (UW) solution (HMP + starch group); modified UW solution (starch omitted) (HMP - starch group) at 0.4 mL/min per g liver; or simply stored in the UW solution (SCS group) at 4 degrees C for 24 hours. Following preservation, livers from HMP + starch, HMP - starch, SCS, and control group (without preservation) were perfused with Krebs-Henseleit Buffer solution at 37 degrees C for 30 minutes. Samples were taken every 10 minutes during 30-minute warm perfusion to assess hepatocyte and endothelial cell function and damage. After 24 hours of hypothermic preservation and 30 minutes rewarming, livers in the HMP + starch group displayed significantly lower lactate dehydrogenase levels and higher bile production. Endothelial cell function was also improved as indicated by hyaluronic acid uptake and shorter transient time for albumin observed in a multiple indicator dilution study. Liver wet and dry ratio and histological findings confirmed reduced edema formation in the tissue of the HMP + starch group livers compared with that of the HMP - starch and SCS group livers. These results suggest that HMP with the UW solution containing starch improve endothelial cell function and induce less hepatocellular damage following 24-hour preservation compared to SCS and HMP with the starch-free UW solution. These results also suggest that oncotic support may be an important component in preserving hepatic microcirculation in HMP.

The potentially detrimental influence of parenchymal cells on endothelial function during preservation in UW solution was examined by co-storage of rat abdominal aortic rings with isolated liver cells. Cold storage of rings in UW solution alone for up to 96 h had no effect on the response to acetylcholine, though constriction was progressively lost. Co-storage of rings with liver cells resulted in no loss of sodium nitroprusside response, but the relaxation response to acetylcholine was reduced. The loss of acetylcholine response could not be attributed to Kupffer cells, the lowering of pH, oxygen depletion, or the loss of constriction. A similar loss of endothelial function was observed in rings stored in pieces of liver, kidney or heart. We conclude that parenchymal cells exude factors during preservation by cold storage which reversibly inhibit vascular NO production. These factors could significantly impair whole organ function on reperfusion.

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor-specific antibodies; all consistent with humoral rejection. These responses were dependent on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) has been implicated in ischemia-reperfusion injury in many tissues under normothermic conditions. The purpose of this study was to determine whether PARP contributes to mechanisms of the hypothermic ischemia-reperfusion injury that occurs when kidneys are cold stored for transplantation. Cortical tissue slice PARP enzyme activity rose significantly with prolonged cold storage and was dependent on both reperfusion and preservation quality. However, prior exposure to warm ischemia abrogated this increase. PARP protein increased with cold storage but was not dependent on reperfusion. PARP enzyme activity rose quickly after reperfusion in buffer and was not different when whole blood was used. Addition of exogenous hydrogen peroxide (3 mM) to normal renal slices significantly increased PARP activity over 4 h in the cortex but not in the medulla, but the medullary basal PARP synthesis rate was five times higher than that in the cortex. However, the reactive oxygen species (ROS) inhibitors catalase (2,000 U/ml), Trolox (200 microM), and DMSO (15 mM) did not reduce reperfusion-induced PARP activity in cold-stored cortical slices. Finally, PARP inhibitors potentiated preservation injury in isolated canine proximal renal tubules. In conclusion, canine renal PARP enzyme activity rises with prolonged cold storage after reperfusion and may play a protective rather than an injurious role in hypothermic preservation for transplantation. ROS are sufficient but not necessary to activate PARP under these conditions.

Background: Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs., Methods: This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival., Results: Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure., Conclusions: These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Background: Donor brain death (BD) has been implicated as a risk factor for the poor performance of kidneys after transplantation in small but not large animal models. This study determined the effects of donor BD on renal function and lipid mediator metabolism in a large animal model of renal hypothermic preservation injury., Methods: Adult beagle donors were subjected to explosive BD for 16 hr. After BD, the kidneys were removed, cold stored for 24 hr in cold University of Wisconsin solution, and allotransplanted into recipient dogs for either 4 hr (group 1) or 7 days (group 2). Controls for both groups consisted of kidneys obtained from living donors. Renal allograft function and tissue arachidonic acid (AA) metabolism were determined after reperfusion., Results: Short-term renal function after transplantation was generally unaffected by BD. Renal blood flow decreased after reperfusion but was not altered during the 16-hr BD period. Neutrophil infiltration significantly increased in kidneys from brain-dead donors before storage and after 4 hr of reperfusion. Renal cortex and medulla AA metabolism were not significantly affected by BD after short-term reperfusion except when thiol-ether leukotrienes (LTC(4)/D(4)/E(4)) were increased with BD. Serum creatinine was elevated during 7 days, but, surprisingly, BD significantly attenuated this injury., Conclusion: BD in large mammals does not significantly affect renal allograft function or AA metabolism after transplantation. The role of BD in human renal preservation injury and inflammation should be reevaluated.

Background: Renal ischemic preconditioning (IPC) is a phenomenon whereby a brief period of ischemia and reperfusion (I/R) provides tolerance to subsequent periods of ischemia. IPC has been demonstrated to protect rodent kidneys during I/R. The applicability to large mammals, including human beings, is unclear. The objective of this study was to determine if renal IPC has a beneficial effect in a large animal model of warm I/R and hypothermic preservation injury, which occurs with renal allografting., Methods: Renal ischemia (45 minutes) and reperfusion was studied in untreated dogs and in dogs receiving IPC (10-minute/10-minute I/R). IPC was administered immediately before I/R (early IPC) or 24 hours before I/R (delayed IPC). In another group of dogs, pharmacologically induced IPC was attempted with local intra-arterial administration of dipyridamole (2.4 mg/kg/min) to increase local adenosine concentrations. Finally, IPC was induced in kidneys before harvest, cold stored for 24 hours in University of Wisconsin flush solution, and subsequently reperfused for 4 hours in allogeneic recipients. Renal functional parameters, including vascular resistance, glomerular filtration rate, urine production, oxygen consumption, and proximal tubular fluid reabsorption, were monitored during the reperfusion period and were compared with the control ischemic group., Results: Renal function significantly declined during I/R, relative to the nonischemic contralateral kidney but was not different with any form of IPC, relative to the ischemic control group not treated with IPC. IPC pretreatment also did not affect the preservation injury observed in cold-stored kidneys reperfused after transplantation., Conclusions: It is concluded that IPC has no significantly measurable effects in warm or hypothermic renal I/R injury in large animals. The clinical usefulness of IPC in human renal ischemic conditions remains uncertain.

Background: Hypothermic machine perfusion preservation has the potential to relieve the current donor shortage problem by reclaiming and preserving marginal donor organs including those from viable non-heart-beating donors. A number of problems exist with the current machine perfusion technology for preserving livers, and much research is needed to determine the clinical impact of this technology in preserving non-heart-beating donor livers., Methods: This study was conducted to compare the poststorage function and microcirculation of simple cold stored and machine perfusion preserved livers that had experienced 30 min of warm ischemia followed by a 10 hr preservation period. In an isolated rat liver perfusion model, lactate dehydrogenase activity, indocyanine green secretion, and portal pressure values were determined at major time points. An intravital microscopy was conducted to assess microcirculation., Results: The results showed an increase in flow homogeneity of machine perfused livers, which correlated with the reduction in portal pressure when compared with simple cold storage (5.4+/-0.4 vs. 8.7+/-0.6 mm Hg). A reduction in lactate dehydrogenase levels in the perfusate (333+/-22 vs.103+/-8 U/L) and an increase in bile production of the machine perfused livers (4.9+/-0.5 vs. 33.2+/-1.7 microg/min/g liver) and indocyanine green secretion (11.7+/-1.7 vs. 21.2+/-2.1 Abs/g bile) were observed at all time points (mean+/-SE of final point given). Intravital microscopic examination indicated that large regions of non flow, as indicated by the absence of fluorescein isothiocyanate-labeled albumin, were observed in the simple cold stored tissue, whereas machine perfused liver showed increase flow homogeneity. Values of bile production, indocyanine secretion, and cellular damages were comparable with controls. Histologic examination confirmed that simple cold stored tissue displayed increased vacuolization, and machine perfused tissue showed regions of normal hepatic structure., Conclusion: These results suggest that machine perfusion for 10 hr improves both poststorage function and microcirculation while reducing cellular damage of liver tissue that has experienced 30 min of warm ischemia, when compared with simple cold storage. Further studies need to be conducted, but this study suggests that machine perfusion preservation has the potential to reclaim and preserve liver tissues after warm ischemic insult.

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.

Background: Brain death has been shown to decrease graft function and survival in rodent models. The aim of this study was to evaluate how brain death affects graft viability in the donor and liver tolerance to cold preservation as assessed by survival in a canine transplant model., Methods: Beagle dogs were used for the study. Non-brain dead (BD) donors served as controls. Brain death was induced by sudden inflation of a subdural balloon catheter with continuous monitoring of arterial blood pressure and electroencephalographic activity. Sixteen hours after confirmation of brain death, liver grafts were retrieved. All livers were flushed in situ and preserved for 24 hr in cold University of Wisconsin solution before transplantation. Recipient survival rates, serum hepatic enzyme levels, coagulation, and metabolic parameters of the recipients were analyzed., Results: No significant changes were observed in serum aminotransferases (alanine and aspartate transaminases) and lactate dehydrogenase levels in the BD donor. After preservation, control (n=6) and BD livers (n=5) showed full functional recovery after transplant with 100% survival in both groups at day 7. There was no significant difference in peak serum alanine, aspartate transaminases, and lactate dehydrogenase after transplantation in recipients who received a liver from BD donor compared to control group. BD livers were functionally as capable as control livers in correcting metabolic acidosis during the first 24 hr posttransplantation. Coagulation profiles (index normalized ratio, activated partial thromboplastin time) after reperfusion were similar between groups., Conclusion: In contrast to previous reports in rodent models, our study shows that brain death does not cause significant liver dysfunction in the donor before organ removal. Donor brain death and prolonged liver graft preservation do not interact significantly to impair liver function and survival after transplantation.

Background: Eighty percent of donor organs come from donors who have suffered brain trauma (brain-dead donors). This unphysiological state alters the hemodynamic and hormonal status of the organ donor. This can cause organ injury, which has been suggested to alter the immunological or inflammatory status of the organ after transplantation, and may lead to increased sensitivity of the organ to preservation/transplantation injury. In this study we asked the question: does brain death cause injury to the liver that decreases successful liver preservation?, Methods: The rat liver transplant model was used to compare survival in rats receiving a liver from a brain-dead donor versus a non-brain-dead donor. Brain death was induced by inflation of a cranially placed balloon catheter. The rats were maintained normotensive with fluid infusion for 6 hr. The livers were flushed with University of Wisconsin (UW) solution and immediately transplanted or cold stored for 20 hr before transplantation., Results: Recipient survival with immediately transplanted livers or those stored for 20 hr was 100% with livers from non-brain-dead donors. However, survival decreased when livers were procured from brain-dead donors. Survival was 75% (6/8) when storage time was 0 hr and 20% (2/10) when the liver was cold stored for 20 hr before transplantation., Conclusion: This study shows that brain death induces alterations in the donor liver that make it more sensitive to preservation/reperfusion injury than livers from donors without brain death. The mechanism of injury to the liver caused by brain death is not known. Because most livers used clinically for transplantation come from brain-dead donors, it is possible that poor function of these livers is due to the intrinsic condition of the donor organ, more than the quality of the preservation. Methods to treat the brain-dead donor to improve the quality of the liver may be needed to allow better preservation of the organ and to give better outcome after liver transplantation.

Isolated rat myocytes cold stored anaerobically up to 24 h in University of Wisconsin solution lost 95% of their ATP and 100% of their glycogen. They underwent contracture when rewarmed in a Krebs-Henseleit (KH) medium that contained Ca unless Ca addition was delayed. In the latter case, cell function, measured by stimulation-induced cell shortening, was surprisingly well retained. Aerobically stored cells were resistant to Ca on rewarming, although 96% of glycogen was still lost, along with 46% of ATP. Cells that were incubated for 48 h aerobically with the substrates glucose and pyruvate at pH 6.2 retained 77% of their ATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, after rewarming, these cells functioned no better than anaerobically stored cells, although their inotropic response to isoproterenol was improved. We conclude that 1) aerobic conditions with substrates at low pH preserve myocyte metabolic reserves well for 48 h, partly by reducing the demand for ATP; 2) rewarming conditions are critical for anaerobically stored cells with metabolic stores that are severely depleted; and 3) unloaded cell function is surprisingly insensitive to a period of severe metabolic deprivation.

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes., (Copyright 2000 Academic Press.)

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solution (4 degrees C) containing [(3)H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis of the ratios of [(3)H] AA and [(14)C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A(2) (PLA(2)) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA(2) activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [micromol phosphorus/L/mg] : 0.197 +/-. 001 at 0 hours) unless energy blockers were used (0.155 +/-.005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/-.001 at 48 hours). This study shows that a net PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA(2) leading to membrane damage.

Background: Calcium plays an important role in liver preservation and preservation induces depletion of cellular Ca. This may affect hepatocyte cytoskeleton integrity necessary for maintaining cell shape and organ viability. We tested the effects of a microtubular stabilizer (Taxol) in liver cell preservation., Methods: Isolated rat hepatocytes were preincubated with or without a microtubule stabilizing agent, 100 microM Taxol, at 37 degrees C for 20 min, then stored in the University of Wisconsin (UW) solution +/-1.5 mM CaC12 at 4 degrees C for up to 48 hr. After storage, the cells were rewarmed in Krebs-Henseleit buffer with air at 37 degrees C for 1 hr. Morphological changes in the plasma membrane (scanning electron microscopy) and cell viability (percentage of lactate dehydrogenase [LDH] release) before and after rewarming were studied., Results: Hepatocytes showed time-dependent increase in bleb formation (cytoskeleton disruption) during cold storage. Rewarming the cells caused even greater bleb formation and increased LDH release (cell death). Pretreatment of cells with Taxol and cold storage in the UW solution with 1.5 mM Ca suppressed both bleb formation and LDH release in 48-hr coldstored cells., Conclusions: Cold storage of hepatocytes leads to reperfusion injury and cell death. This can be suppressed with Taxol and Ca. This suggests that hypothermia induces changes in cellular Ca and a disruption of the microtubules, leading to loss of cell viability. Improved liver preservation may require suppression of Ca-dependent disruption of the cytoskeleton system of liver cells.

Background: A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated., Methods: Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine)., Results: There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis., Conclusion: The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.

Background: A limitation to fully using lung transplantation for patients with end-stage lung diseases is short, safe preservation time (4 to 6 hours). Our goal is to extend this to 24 hours or more, which would greatly improve clinical lung transplantation., Methods: We used the isolated perfused rat lung to test how two preservation solutions (low potassium dextran and University of Wisconsin solution) affected quality of lungs after 6, 12, and 24 hours of preservation. Also, we tested modifications of the University of Wisconsin solution, including reversing the ratio of Na/K, the addition of 1.5 mmol/L calcium, and the combination of calcium and butanedione monoxime, agents that improve cardiac preservation. After preservation at 4 degrees C, lungs were reperfused at 37 degrees C with a physiologically balanced solution. Pulmonary artery flow rate, airway peak inspiratory pressure, and tissue edema were used to assess degree of preservation and reperfusion injury., Results: Low potassium dextran solution gave poor preservation (decreased pulmonary artery flow, tissue edema) after 12 hours of cold storage. There were no differences between regular and reversed Na/K ratio University of Wisconsin solutions at 12 or 24 hours of preservation. Addition of calcium had no beneficial effect on lung preservation. However, University of Wisconsin solution with calcium and butanedione monoxime gave excellent 24-hour cold storage, with pulmonary artery flow rate, tissue edema, and airway peak inspiratory pressure equal to control (0 hours of preservation) lungs., Conclusions: The University of Wisconsin solution appears capable of lung preservation for up to 24 hours if modified to contain calcium and butanedione monoxime. The mechanism of action of butanedione monoxime may be related to the suppression of smooth muscle contraction resulting in vasodilation of the cold-stored lung on reperfusion.

Background: Preservation injury is multifactorial and its mechanism is still incompletely defined. Calcium may play an important role in preservation injury., Methods: The effects of hypothermia on cytosolic free calcium concentration ([Ca2+]I) and total cellular calcium content in isolated rat hepatocytes were investigated by using fura-2 fluorescence and atomic absorption spectroscopy. Fura-2 loaded cells were placed into a prechilled (7 degrees C) cuvette equipped with a stirrer or preserved in the University of Wisconsin (UW) solution for up to 48 hr. In some experiments, cells were pretreated with inhibitors of Ca2+ release from mitochondria (m-iodobenzylguanidine [MIBG]) and from endoplasmic reticulum (ryanodine [RYA]) for 20 min at 37 degrees C. Mitochondrial functions after preservation were evaluated by measuring ATP and respiratory rates., Results: Cooling to 7 degrees C caused a rapid increase in [Ca2+]I that was substantially blocked by MIBG and RYA pretreatment. The elevated calcium gradually leaked out of the cells into the Ca2+-free medium. In long-term storage of the cells in the UW solution, there was a marked decrease in both cytosolic free calcium and total cellular calcium. Pretreatment of the livers with MIBG before cold preservation in the UW solution resulted in a stimulation of ATP regeneration in tissue slices. MIBG pretreatment also improved mitochondrial respiratory functions after cold preservation., Conclusions: Thus, the loss of mitochondrial function after liver preservation in the UW solution may be related to the effects of hypothermia on calcium metabolism. Approaches to help maintain calcium homeostasis during storage may improve organ preservation.

Metabolic suppression by temperature is a key to successful organ preservation. Additional methods for inducing metabolic suppression may further improve organ preservation. Extracellular acidosis has been shown to suppress warm anoxic injury to various isolated cells. Acidosis may suppress enzymes with a pH optimum at the pH of the cytosol (pH 7.3). In this study, the combination of hypothermia and acidosis was used to determine if it would improve renal preservation. Dog kidneys were cold-stored (CS) for 48 h in University of Wisconsin (UW) solution with the pH adjusted to 6.4, 6.8, 7.4, or 7.8. Kidneys were also machine-perfused (MP) for 3 days with the gluconate perfusion solution (Belzer's machine perfusion solution, MPS) at pHs similar to those tested for CS. Renal function (serum creatinine, SCr) and survival were recorded in immediate contralateral nephrectomized recipients. On the basis of maximum SCr values, kidneys preserved by CS or MP were best preserved at pHs of 7.4 or 7.8. At a pH of 6.8, SCr values were elevated and returned to normal at a slower rate than in those preserved at higher pHs. This study shows that acidosis is not cytoprotective to cold-stored dog kidneys and causes preservation/reperfusion injury.

We have previously shown that livers from long-term-fasted rats acquire tolerance to warm ischemic injury following transplantation, despite the fact that fasting depletes glycogen and ATP from the liver. The precise mechanism of the protective effect induced by donor fasting, however, is still a matter of controversy. In this experiment we determined heat-shock protein (GRP78) mRNA expression in livers during long-term fasting and TNF-alpha mRNA expression in transplanted livers exposed to warm ischemia. We also measured the concentration of TNF-alpha by ELISA in the ascitic fluid of fed and fasted rats injected intraperitoneally with zymosan to investigate why livers from fasted rats tolerate ischemic injury better. There seemed to be a positive correlation between GRP78 mRNA expression and survival. TNF-alpha secretion into the ascitic fluid of fasted rats was markedly suppressed, and fasting donor animals induced cytoprotective substances, such as GRP78, in the liver. These factors may contribute to the tolerance to ischemic injury produced by donor fasting.

The purpose of this study was to compare the effects of the University of Wisconsin solution plus butanedione monoxime, the University of Wisconsin solution without butanedione monoxime, and saline on the preservation of muscle tissue. Forty-nine rat hindlimbs were amputated and replanted. The study population was subdivided into four groups according to flushing solution, storage, and replantation protocols. The limbs of the control group (n = 12) were flushed with 20 ml University of Wisconsin solution and immediately replanted onto the same rat. In the remaining three groups, the limbs were immersed in solution, stored in a refrigerator at 4 degrees C for 24 hours, and then replanted onto a fresh rat. The limbs in the no flushout group (n = 7) were placed into storage in cold saline solution without being flushed. The limbs in the University of Wisconsin solution group (n = 17) were flushed with 20 ml of University of Wisconsin solution prior to storage, and those in the University of Wisconsin solution plus butanedione monoxime group (n = 13) were flushed with 20 ml University of Wisconsin solution plus 20 mM butanedione monoxime. Limb survival rate was 100 percent for the control and University of Wisconsin solution plus butanedione monoxime groups, 87 percent for the University of Wisconsin solution group, and 71 percent for the no flushout group. Seven days after replantation, ATP levels were 71 percent of control in the University of Wisconsin solution plus butanedione monoxime group, 33 percent in the University of Wisconsin solution group, and 29 percent in the no flushout group. Tissue K+/Na+ ratio showed that the University of Wisconsin solution plus butanedione monoxime group maintained electrolyte balance, whereas the balance was significantly lowered in University of Wisconsin solution and no flushout groups. The University of Wisconsin solution plus butanedione monoxime limbs did not exhibit cell swelling, whereas total tissue water values for the University of Wisconsin solution and no flushout groups increased significantly. Serum creatinine kinase, measured 24 hours after replantation, was 120 percent of control in the University of Wisconsin solution plus butanedione monoxime group, 550 percent in the University of Wisconsin solution group, and 772 percent in the no flushout group. Limbs in the University of Wisconsin solution plus butanedione monoxime group had more flexible ankle joints and pliable muscle (i.e., less contracture) than those in the University of Wisconsin solution and no flushout groups. In conclusion, rat hindlimbs can be preserved hypothermically for 24 hours using the University of Wisconsin solution, the University of Wisconsin solution plus butanedione monoxime, or saline. However, the University of Wisconsin solution plus butanedione monoxime limbs had better ATP levels and less cellular injury after replantation. Based on these results, we believe that, biochemically, flushing and storage of muscle tissue in the University of Wisconsin solution plus butanedione monoxime are the most effective means of those studied for preserving composite tissue grafts for 24 hours.

Liver cold storage leads to oxygen free radical production and reperfusion injury. Antioxidants are effective in suppression reperfusion injury in rat livers when used in the reperfusion medium. However, in clinical liver transplantation their effectiveness is not clear, which may be due to the way they are used (in the recipient). In this study we compare the effectiveness of antioxidants when used in the reperfusion medium versus the cold storage solution in isolated hepatocytes and the isolated perfused liver. Hepatocytes were cold stored in UW solution for 24 h. Oxidative stress, induced by t-butyl hydroperoxide (tBHP), was measured in the presence of one of five different antioxidants--deferoxamine (DFO), dithiothreitol (DTT), trolox, tocopherol, dimethylthiourea (DMTU)--in the reperfusion buffer or UW solution. Efficacy was judged by reduction in membrane damage (LDH release) during rewarming. Also, rat livers were cold stored for 48 h in UW solution (+/- antioxidant) and reperfused (+/- antioxidants). Efficacy was judged by the effect on enzyme release and bile production. Cold storage of hepatocytes for 24 h sensitized them to oxidative stress. The concentration of tBHP required to induce maximal cell death (80%-90% LDH release) was reduced from 1.3 mM (fresh cells) to 0.37 mM (LD-50 values). All antioxidants except DMTU suppressed oxyradical-induced LDH release when used in the reperfusion medium, but only DFO was effective when used in the UW solution. In the isolated perfused liver, DFO, DTT, and trolox were effective and suppressed enzyme release when added to the reperfusion buffer, but none were effective when used in the UW solution. We conclude that cold storage sensitizes liver cells to oxidative stress. The most effective antioxidant was the iron chealator, DFO, which was effective in the reperfusion buffer (isolated perfused sliver or hepatocytes) but not in the UW solution when tested in the isolated perfused liver. Suppression of reperfusion injury in liver transplantation could be obtained by antioxidant therapy. However, it is unclear how best to deliver the antioxidants to the site of oxyradical generation.

In this study, we tested the effect of donor fasting with or without the use of an essential fatty acids deficiency (EFAD) diet in the recipient using rat heart, pancreas, and liver transplant models. We then compared the survivals, tumor necrosis factor alpha (TNF-alpha) response, and white cell accumulation in rats in order to clarify the mechanisms of the beneficial effect of donor fasting and recipient EFAD. It was found that when the grafts were obtained from fasted donors and then transplanted into fed recipients, the survival rate was significantly higher for all three grafts than for those obtained from fed rats and transplanted into fed rats. The best survival was seen for pancreas grafts obtained from fasted donors and then transplanted into EFAD recipients. TNF-alpha secretion was significantly suppressed in both fasted and EFAD rats, and both the total cell count and neutrophil count were suppressed in EFAD rats. These results clearly indicate that in addition to liver grafts, both heart and pancreas grafts obtained from fasted animals are more tolerant to warm ischemic injury. Furthermore, the combination of donor fasting and recipient EFAD acts synergistically to inhibit the post-transplantation inflammatory reaction (through decreased TNF-alpha secretion and white cell accumulation), thus resulting in an improved survival.

In this study we investigated the effect of calcium addition to the UW solution on the quality of the preserved rat liver as judged by normothermic isolated perfusion. Rat livers were cold stored in UW solution containing varying concentrations of calcium chloride (0, 0.5, 1.5, 5.0 mM) for periods of 0, 24 and 48 hours. At the end of the preservation period the livers were reperfused for 90 minutes at 37 degrees C with Krebs Henseleit Buffer. The quality of preservation was assessed by quantification of enzyme release, bile production and protein synthesis. The addition of 1.5 mM calcium to the UW solution suppressed the incidence of damage in the 24 hour cold stored liver similar to control livers (0 hours preserved). LDH release were significantly reduced from 22.1 +/- 7.3 (units/hr/g) in regular UW to 9.4 +/- 0.8 (units/hr/g) in UW plus 1.5 mM calcium. AST release also was suppressed by the addition of calcium to the UW. Bile production was enhanced by the addition of calcium; from 21.3 +/- 0.6 (mg/hr/g) in regular UW to 46.3 +/- 5.9 (mg/hr/g) in UW plus 1.5 mM calcium. Protein synthesis was reduced to 38% of control after 24 hr cold storage and was unchanged by the addition of calcium to the preservation solution. Although the addition of calcium to the UW solution improved the preservation of the 24 hour cold stored liver it did not offer the same degree of protection to the 48 hour preserved liver. Therefore, calcium addition may be one agent for improving preservation for short term cold storage of the liver but longer term storage will require other modifications as well.

Simple cold storage of livers for transplantation activates glycolysis due to lack of oxygen. Energy derived from glycolysis may be critical for cell survival and liver cell death may occur once glycolysis is inhibited in the liver due to accumulation of end products or lack of substrates (glycogen). The relationship between cell death (lactate dehydrogenase, LDH release), anaerobic glycolysis (lactate production), and glycogen content of liver tissue was studied during cold incubation of liver slices in UW solution. Rat livers slices from male Sprague Dawley rats were incubated at 4 degrees C in UW solution, with continuous gentle shaking, under conditions of chemical hypoxia (KCN, 5 mM). The rate of lactate production, LDH release-ATP and glycogen content were measured spectrophotometrically and by HPLC. Lactate increased nearly linearly for the first 48 h of incubation; total lactate which had accumulated after 48 h was 33.9 +/- 0.81 mumol/g and at 96 h nearly the same, 31.3 +/- 1.2 mumol/g. Glycolysis stopped, apparently, because of the depletion of liver slice glycogen which was initially 228.8 +/- 1.7 mumol/g wet wt. It decreased to 34.7 +/- 2.7 mumol/g at 48 h and to 18.7 +/- 1.1 mumol/g at 72 h and remained at this level for the next 24 h. An increased leakage of LDH occurred once glycogen metabolism (and accumulation) ceased. LDH release could be stimulated after only a few hours of cold incubation of liver tissue slices by adding glycolysis inhibitor (iodoacetic acid) to the medium. After 24 h. LDH release was 24.4 +/- 1.8% and increased to 52.8 +/- 5.2% (P < 0.05, Student's t-text) with iodoacetic acid. Adding a glycolytic substrate (fructose, 10 mM) to the medium maintained lactate production for 96 h. The stimulation of glycolysis by fructose also reduced cell death: LDH release was significantly lower at 72- and 96-h incubation (P < 0.001, two-way ANOVA). The ATP content was significantly higher with fructose (P < 0.001). Adding glucose (20 mM) and fructose (10 mM) in combination resulted in prolonged cell survival, significantly delayed glycogen depletion and significantly higher ATP content at 48 and 72 h (two-way ANOVA). Livers from rats who had fasted for 24 h demonstrated the same LDH release at 48 h when incubated with glucose (20 mM) and fructose (10 mM). In conclusion, LDH leakage from hypoxic cold-stored liver slices is related to anaerobic glycolysis. Anaerobic glycolysis appears to continue slowly under hypothermia and provides sufficient energy for maintenance of cell viability. A stimulation of glycolysis in the cold is possible by fructose and results in prolonged cell survival under hypothermic conditions. Glycogen depletion can be slowed down by combining glucose and fructose.

Livers from fasted animals are believed to be more vulnerable to ischemic injury than those from fed donors. However, we have recently shown the opposite: livers from fasted rats were more tolerant to ischemic injury. Indeed, the survival rate of 60 min warm ischemic damaged livers increased from 0 to 90% if donor rats were fasted for three days. In this study, we examined how donor fasting affects the outcome of pancreas and heart preservation. BN rats were used as both donors and recipients, and recipients of pancreatic grafts were rendered diabetic prior to transplantation. Pancreatic or heart grafts were subjected to 90 min or 25 min of warm ischemia and were transplanted into the right side of the necks of recipients rats. The viability rate of hearts transplanted from fed donors into fed recipients was only about 11% (1/9) after transplantation. However, the viability rate with fasted donors was 75% (6/8). The rate of successful pancreatic grafting from fed donors into fed recipients was 28.6% (2/7), and that from fasted donors to fed recipients was 41.7% (5/12). These results confirm that the nutritional status of the donor is an important factor in the outcome of not only liver, but also pancreas and heart preservation during transplantation, although the effect of fasting on pancreatic graft is marginal.

Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs-Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed < 15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in < 30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 +/- 5.1%; TS-A, 52.9 +/- 0.8%, TS-B, 53.6 +/- 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 +/- 1.8 and 26.3 +/- 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell-cell relationship.

Altered cellular calcium (Ca) homeostasis may be important in mediating hypothermic injury in preserved kidneys. In this study the effect of hypothermic (5 degrees C) storage on ionized intracellular Ca concentration ([Ca]i) in rabbit tubules was examined using Indo-1. Tubules were stored up to 250 min in UW-gluconate solution containing either 0.0, 0.5, 1.5, or 5.0 mM Ca (yielding about 3.6, 62, 371, and 1,010 microM ionized solution Ca (Ca2+) at 5 degrees C, respectively). [Ca]i increased to about 1,600 nM within 1 min after suspension in UW solution followed by a decrease in [Ca]i during the subsequent 60 min in all groups, suggesting mitochondrial Ca sequestration. Thereafter, [Ca]i either 1) increased in tubules incubated with 1.5 and 5.0 mM Ca to levels greater than 2,500 nM; 2) decreased to about 800 nM in tubules incubated with 0.5 mM Ca and then remained stable; or 3) continued to decrease in tubules incubated with 0.0 mM added Ca to reach an apparent steady-state concentration of about 175 nM after 180 min of incubation. The early spike in [Ca]i was unaffected by adding EGTA (solution Ca2+ = 50 nM). Ryanodine eliminated the [Ca]i spike, indicating that cooling in UW-gluconate solution caused release of endoplasmic reticulum Ca. This study shows that [Ca]i initially increases after exposure to UW-gluconate solution and appears to be transiently buffered through intracellular, probably mitochondrial, sequestration. Saturation of cellular buffer mechanisms resulted in a sustained dependence of [Ca]i on extracellular Ca2+. These results support the hypothesis that the effect of Ca on kidney viability is related to solution-induced alterations in [Ca]i.